In vitro modelling of human proprioceptive sensory neurons in the neuromuscular system

Proprioceptive sensory neurons (pSN) are an essential and undervalued part of the neuromuscular circuit. A protocol to differentiate healthy and amyotrophic lateral sclerosis (ALS) human neural stem cells (hNSC) into pSN, and their comparison with the motor neuron (MN) differentiation process from the same hNSC sources, facilitated the development of in vitro co-culture platforms. The obtained pSN spheroids cultured interact with human skeletal myocytes showing the formation of annulospiral wrapping-like structures between TrkC + neurons and a multinucleated muscle fibre, presenting synaptic bouton-like structures in the contact point. The comparative analysis of the genetic profile performed in healthy and sporadic ALS hNSC differentiated to pSN suggested that basal levels of ETV1, critical for motor feedback from pSN, were much lower for ALS samples and that the differences between healthy and ALS samples, suggest the involvement of pSN in ALS pathology development and progression.

. Gene expression (ΔCt) of target genes at TP1 and TP3. Boxplots with Tukey whiskers represent the gene expression levels obtained in healthy (white) and ALS (grey) samples before any differentiation protocol (at TP1) and with different protocols at TP3. Each value from the graph is obtained normalising Ct raw values against the housekeepers (B-ACTIN, GAPDH, RPS18S) and calculating the average. Higher Ct values indicate later amplification and therefore lower gene expression level. For each gene and protocol, the results of Mann-Whitney test against TP1 results are indicated above each bar with stars. P-values (*<0.05; **<0.01; ***<0.001; ****<0.0001). Boxplots with Tukey whiskers represent the gene expression levels obtained in samples of the gene expression of each housekeeper at TP1 and TP3 with different protocols, clustered in healthy (white) and ALS (grey) samples. Each value from the graph represents obtained raw Ct values. Outliers are shown with a triangle shape. Mann-Whitney statistics test was conducted for unpaired samples, resulting in non-significant differences between ALS and healthy samples.

Fig. S4. Characterisation of spheroids undergoing SN differentiation protocols at TP2. A)
Floating spheroid immunostaining at TP2. Maximum z-projection of fluorescence images obtained of the immunostaining of healthy samples undergoing SN differentiation protocol-A at TP2. DAPI is shown in blue, HNK1 in green, and TUJ1 in red. Scale bar 100µm. B) Flow cytometry analysis of HNK1 on 2D culture of healthy (orange), 2D cultures of ALS (green), and 3D spheroids of healthy samples at different densities (red), all undergoing Protocol A. C) Flow cytometry analysis of HNK1 of healthy spheroids undergoing protocols A' and D (without CHIR99021, in orange), healthy spheroids undergoing protocols B and C (with CHIR99021, in green), ALS spheroids undergoing protocols A' and D (without CHIR99021, in red), and ALS spheroids undergoing protocols B and C (with CHIR99021, in purple). In all FC graphs, negative control of non-stained healthy samples is shown in blue.  Timepoints are indicated referring to the days of differentiation (ddiff). A) Bright field images of floating spheroids of three different initial seeding densities of hNSC (3.000, 4.500 and 6.000 cell · spheroid −1 ) undergoing pSN differentiation protocol A or MN differentiation protocol. Images taken at different days after differentiation started (ddiff). Scale bar 100 µm. B) Quantification of spheroid area of floating spheroid images at different timepoints. The box and whiskers plots represent the median values and interquartile ranges for measurements of the three seeding densities obtained for MN and SN spheroids; whiskers represent maximum and minimum values obtained; (n=3, Mann-Whitney test). C) Analysis of all measurements grouped obtained for MN and SN spheroids; bars represent mean ±SD of all values obtained; t-test for unpaired samples with Welch's correction (not assuming equal SDs) resulting in a p-value of 0.0038, **p-value<0.01.

Fig. S7. Comparison of hNSC healthy spheroids differentiated towards pSN and MN as platted spheroids.
Timepoints are indicated referring to the days of differentiation (ddiff). Differentiation of healthy hNSC as individual or adjacent plated spheroids. Bright field images of the differentiation of hNSC to SN through differentiation protocol A or to MN. Images taken at different timepoints (n=2). Scale bar 100 µm.

Sample of study Highlights
Ref.
Rat primary sensory neurons cocultured with rat primary myocytes Immunofluorescence analysis revealed the presence of annulospiral wrapping (ASW) and flower spray ending (FSE), together with the expression in sensory terminals of the stretch sensitive sodium channel BNaC1 and the membrane support protein PICK1.
Calcium currents imaging after stretching an intrafusal muscle fibre through microelectromechanical systems, cantilever deflection, detected the presence of physiologically relevant sensory endings.  Table S1. Summary of the publications studying the sensory afferent pathway in vitro in the last decade though SN-SkM coculture. The cells utilised and highlighted results are described for each publication reference.

Gene
Encoding protein and function GAPDH Glyceraldehyde 3-phosphate dehydrogenase plays a role in cell glycolysis and nuclear functions. It is implicated in metabolic and non-metabolic functions, such as initiation of apoptosis or axoplasmic transport. It is frequently used as a housekeeper gene.
ACTB β-actin, a human isoform of actin, is a highly preserved protein involved in cell structure, integrity and motility. It is frequently used as a housekeeper gene.

RPS18
Ribosomal protein S18 is a component of the 40S subunit of ribosomes, organelles that catalyse protein synthesis. It is frequently used as a housekeeper gene.

HNK1
Galactosylgalactosylxylosylprotein 3-β-glucuronosyltransferase 1 (B3GAT1) is an enzyme that in humans is encoded by the B3GAT1 gene, also known in immunology as HNK1 (human natural killer-1). It is involved in cell metabolism and expressed in NCSC. 46 It is known to be upregulated in ALS. 56 POU4F1 POU domain, class 4, transcription factor 1 (POU4F1) also known as brain-specific homeobox/POU domain protein 3A (BRN3A), is a protein highly expressed in the developing peripheral sensory nervous system (e.g. DRG). 35,38,93

NTRK2
Tropomyosin receptor kinase B (TrkB), also known as neurotrophic tyrosine kinase receptor type 2 (NTRK2) is expressed in some mechanoreceptive type of sensory neurons and binds to brain-derived neurotrophic factor (BDNF). 37,61

NTRK3
Tropomyosin receptor kinase C (TrkC), also known as neurotrophic tyrosine kinase receptor type 3 (NTRK3), is expressed in proprioceptive type of sensory neurons and some mechanoreceptive neurons. 61 It binds to neurotrophin-3 (NT-3), mediating neuronal differentiation and survival. 37,94 RUNX3 Runt-related transcription factor 3 (Runx3) regulates survival and axonal projections of proprioceptive sensory neurons. 60 Runx3 has a different regulation in each subtype of TrkC+ neuron. 67 DICER Dicer is essential for maintenance rather than initiation of synaptic contacts in sensorimotor connections, and for processing micro RNAs (miRNAs). Selective impairment in sensory neurons causes sensory ataxia. 59,60 ETV1 ETS variant 1 expression is induced by NT3-TrkC signalling and it is involved in proprioceptive axon projection regulation. 60,66 It is also needed for the survival and differentiation of pSN. 95 But it is also critical in the formation of functional connections between pSN and MN. 66

SPP1
Secreted phosphoprotein 1 is important for cell-matrix interaction and immune functions, and is known to be expressed in pSN. 61 It is frequently present in the inflammatory environment of dystrophic and injured muscles. 61,62

VGluT1
Vesicular glutamate transport 1 (VGluT1), also known as solute carrier family 17 member 7 (SLC17A7), transports glutamate to synaptic vesicles before exocytotic release. It is expressed in pSN at the site of innervation of intrafusal fibers. 96 But it is also detected at the synaptic contacts of interneurons coming from the spinal cord and descending cortical axons, being expressed in the spinal cord in the dorsal horn, intermediate gray and ventral horn, and in sensory DRG neurons. 26 PVALB Parvalbumin, although frequently used as a proprioceptive sensory neuron marker, is also expressed in other cells of the motor system, including but not limited to motoneurons and interneurons within the spinal cord and muscles. 59,65,66 MBP Myelin basic protein is the major constituent of the myelin sheath formed by oligodendrocytes, in the CNS, and Schwann cells, in the PNS. Both MN and pSN are myelinated cells. 61 Anormal levels of MBP are related with demyelinating diseases, multiple sclerosis and ALS. Myelin sheath ultrastructure is known to be disorganized in ALS. 97 Furthermore, oligodendrocyte maturation and MBP expression is known to be reduced in ALS, 10,11 and myelinating Schwann cells are known to be damaged in ALS and trigger inflammatory mechanisms in ALS peripheral nerves. 5,6 GFAP Glial fibrillary acidic protein is one of the major filament proteins of mature astrocytes. It is also present in Schwann cells under stress conditions and following nerve damage or denervation, and it has been found to be upregulated in the peripheral nerve Schwann cells in ALS. 57

MNX1
Motor neuron and pancreas homebox 1, also known as homeobox HB9 (HLXB9), used for identification of MN, it is involved in neural stem cell differentiation pathway and lineage-specific markers.

TNFSF1B
Tumour necrosis factor receptor superfamily member 1B, also known as p75 tumour necrosis factor receptor (P75TNFR), mediates anti-apoptotic and inflammation signals, and it is related with ALS pathway as it is observed in higher expression levels in ALS patients. 58 Table S2. Genes analysed in the qPCR: gene name. encoding protein and function. The information for gene description is taken from the databases Genecards 98 and OMIM, 99 and other papers cited below.